Project Summary/Abstract Obesity is a highly prevalent and costly disease, but effective and non-invasive treatment options are severely limited. The amylin system is considered a promising candidate for the development of novel anti- obesity pharmacotherapies, as amylin agonists decrease food intake and produce weight loss in humans and animal models. The ventral tegmental area (VTA), a mesolimbic nucleus with critical roles in feeding and motivated behavior, is a physiologically and pharmacologically relevant site of action for the energy balance effects of amylin. However, the mechanisms underlying VTA amylin receptor-mediated hypophagia remain unresolved. In particular, the impacts of key variables like sex and diet on the ability of VTA amylin receptor activation to promote negative energy balance are unknown. The overarching goal of this proposal is to identify novel neuroanatomical and behavioral mechanisms by which VTA amylin receptor signaling promotes hypophagia and weight loss, with particular focus on the role of sex and diet in these effects. This award will facilitate the applicant's advancement toward becoming a fully independent PI by exploring new topics in mesolimbic amylin signaling and generating crucial preliminary data for a future R01 application. Specific Aim I uses an innovative combination of virogenetics and behavioral pharmacology to test whether stimulation of VTA-projecting neurons in the lateral hypothalamus, a central source of amylin, activates VTA amylin receptors to control energy balance. This will expand our knowledge by investigating the inputs to VTA amylin receptors, and will provide insight into sex and diet differences by testing male and female rats maintained on chow or high-fat diet (HFD). Specific Aim II evaluates the physiological relevance of VTA amylin receptors for long-term energy balance control in females by testing the feeding and body weight effects of VTA amylin receptor knockdown. This Aim also examines the phenotype of VTA amylin receptor-expressing cells in females with a combination of immunohistochemistry and in situ hybridization. The studies described in this application build upon the applicant's current K01 award by investigating the neural circuits and behavioral mechanisms by which VTA amylin receptor signaling controls energy balance, and by addressing dietary influences (e.g., palatable HFD or bland chow) on the VTA amylin system. These studies are also distinct from the aims of the K01 by exploring a novel and important topic: the role of sex differences in mesolimbic amylin signaling. These studies are designed to produce informative data that will serve as a generative starting point for follow-up questions to be addressed in an R01 application. The novel combination of in vivo and ex vivo techniques used in the proposed studies will further our understanding of the distributed actions of amylin in the brain for energy balance control, filling important gaps in our scientific knowledge that may further the development of novel amylin-based pharmacotherapies for the treatment of obesity.